International Journal of Molecular Sciences Article Enantiocontrolled Preparation of Υ-Substituted Cyclohexenones: Synthesis and Kinase Activity Assays of Cyclopropyl-Fused Cyclohexane Nucleosides Sergio Jurado 1 , Beatriz Domínguez-Pérez 1 , Ona Illa 1 , Jan Balzarini 2 , Félix Busqué 1, * and Ramon Alibés 1, * 1 Departament de Química, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 2 Laboratory of Virology and Chemotherapy, Rega Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium * Correspondence: felix.busque@uab.cat (F.B.); ramon.alibes@uab.cat (R.A.) Abstract: The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone deriva- tives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile inter- mediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a–e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs. Keywords: asymmetric synthesis; carbocyclic nucleosides; HSV-1 thymidine kinase; enzymatic assays Citation: Jurado, S.; Domínguez- Pérez, B.; Illa, O.; Balzarini, J.; Busqué, F.; Alibés, R. Enantiocontrolled Preparation of Υ-Substituted Cyclohexenones: Synthesis and 1. Introduction Kinase Activity Assays of Optically active Υ-substituted cycloalkenones are compounds of synthetic importance Cyclopropyl-Fused Cyclohexane that are used as precursors in the synthesis of natural products and pharmaceutically Nucleosides. Int. J. Mol. Sci. 2022, 23, active molecules [1–4]. In particular, both enantiomers of 4-hydroxy-2-cyclohexenone 1a 9704. https://doi.org/10.3390/ and their O-protected derivatives, such as 1b,c, have been extensively used in organic ijms23179704 synthesis (Figure 1) [5–7]. These chiral cycloalkenones have been prepared by different Academic Editor: Istvan Simon methodologies that involve enzymatic transformations [8], kinetic resolutions [9], chiral Int. J. Mol. Sci. 2022, 23, x FOR PEER auxiliaries REVIEW [10], asymmetric catalysis [11], or chiral pool compounds [12,13]. However,2 most of 15 Received: 22 July 2022 of these approaches do not provide access to both enantiomers. Therefore, the development Accepted: 23 August 2022 of efficient routes to such relevant building blocks is of considerable interest. Published: 26 August 2022 O Publisher’s Note: MDPI stays neutral B O with regard to jurisdictional claims in OR published maps and institutional affil- iations. O O O O O Ph H OH H Ph Copyright: © 2022 by the authors. 1 2 3 4 Licensee MDPI, Basel, Switzerland. a, R = H a, B = A This article is an open access article b, R = TBS b, B = G distributed under the terms and c, R = Bn c, B = U conditions of the Creative Commons d, B = T Attribution (CC BY) license (https:// e, B = C creativecommons.org/licenses/by/ Figure 1. Chiral Figure 1. Chiral cyclohexenone cyclohexenone building blocks 1a–c, nucleoside nucleoside analogues 2a–e built on on aa bicyclo bicyclo 4.0/). [4.1.0]heptane scaffold [4.1.0]heptane scaffold and and synthetic synthetic precursors precursors33and and4. 4. In order to perform these enzymatic assays, to complete the 5′-hydroxymethylbicyclo [4.1.0]heptanyl family 2 by preparing the cytosine analogue 2e, and considering our work Int. J. Mol. Sci. 2022, 23, 9704. https://doi.org/10.3390/ijms23179704 https://www.mdpi.com/journal/ijms on the synthesis of bioactive products bearing a cyclohexane unit [16–18], general and easy access to both enantiomers of 1a and 1b was required. We envisaged that the Int. J. Mol. Sci. 2022, 23, 9704 2 of 15 In a previous work, we reported the synthesis of the nucleoside analogues (NAs) 2a–d built on a bicyclo [4.1.0]heptane scaffold, starting from cyclohexenone 3 bearing a dihydrobenzoin moiety as the chiral auxiliary [14]. These NAs were designed as potential antiherpetic agents by a molecular modeling study on the HSV-1 TK active site that is involved in the first phosphorylation step of the activation process. However, none of the compounds showed significant antiviral activity at subtoxic concentrations (~250 µM). The lack of activity indicated that these functionalized NAs might, eventually, fail to reach the HSV DNA polymerase interaction step. Therefore, further studies were needed to identify the molecular basis of the antiherpetic inactivity of the prepared compounds; in particular, single enzymatic assays were required to test the suitability of these compounds to pass the usual rate-limiting first phosphorylation step [15]. In order to perform these enzymatic assays, to complete the 50 -hydroxymethylbicyclo [4.1.0]heptanyl family 2 by preparing the cytosine analogue 2e, and considering our work on the synthesis of bioactive products bearing a cyclohexane unit [16–18], general and easy access to both enantiomers of 1a and 1b was required. We envisaged that the asymmetric transfer hydrogenation (ATH) on 1,4-cyclohexenedione monoethylene ketal 4 could be applied to prepare such chiral cyclohexenones [19]. ATH with bifunctional ruthenium cata- lysts has become one of the most practical and versatile tools for accessing enantiomerically enriched alcohols in organic synthesis, due to its excellent selectivity, practical simplicity, and wide substrate scope [20,21]. In this article, we disclose a concise entry to both enan- tiomers of Υ-hydroxycyclohexenone by ATH via catalyst control, their use as precursors in the preparation of the conformationally restricted cytosine analogue 2e, and the results of the enzymatic affinity of compounds 2a–e on HSV-1 thymidine kinase. 2. Results and Discussion The synthesis of both enantiomers of 1a and 1b (Scheme 1) started with enone 4, which was easily prepared on a multigram scale from the commercially available 1,4- cyclohexanedione monoethylene acetal in 88% yield, through a two-step optimized de- hydrogenation protocol that involved the formation of the corresponding silyl enol ether followed by oxidation with IBX·MPO complex [17]. The asymmetric reduction of enone 4 was first carried out in a biphasic medium (CH2 Cl2 /H2 O 1:1), using (R,R)-Noyori-I, (R,R)-5 as catalyst, TBAC as a phase-transfer agent, and HCOONH4 as the hydrogen source [22–24]. Under these experimental conditions, allylic alcohol (R)-6 was obtained, along with its totally hydrogenated derivative 7 in a 6:1 ratio and 67% yield. The reduction also resulted in the removal of the ketal protecting group, providing variable amounts of 1a. However, when the reaction was performed using HCOONa [25], the desired allylic alcohol (R)-6 was isolated in 79% yield. Next, deprotection of the carbonyl group was achieved by treatment with montmorillonite K-10 in dichloromethane at room temperature, affording the volatile 4-hydroxy-2-cyclohexenone (R)-1a in 64% yield and 92% ee (determined by CHPLC). Pro- tection of the alcohol 4 as a silyl ether with imidazole and TBSCl in dichloromethane and subsequent hydrolysis of the ketal using montmorillonite K-10 led to (R)-1b in 87% yield for the two steps. In a similar way, the asymmetric reduction was performed with the enantiomeric (S,S)-5 catalyst, providing allylic alcohol (S)-6 in 78% yield, along with traces of the totally hydrogenated derivative. Hydrolysis of the ketal delivered (S)-1a in 62% yield and 92% ee (determined by CHPLC), while silyl ether protection and ketal cleavage afforded (S)-1b in 88% yield. Thus, we established a practical enantioselective procedure to prepare both enantiomers of 4-hydroxy-2-cyclohexenone (R)- and (S)-1a in four steps, ca. 44% overall yield and 92% ee, and their O-silyl derivatives, (R)- and (S)-1b, in five steps and 60% overall yield, which compared with other published methodologies [5–13]. Both enantiomers of the product could be obtained from the same precursor only by selecting either the (S,S)- or (R,R)-5 catalyst. Int. Int. J.J. Mol. Mol. Sci. 2022, 23, Sci. 2022, 23, 9704 x FOR PEER REVIEW 33 of 15 15 Scheme 1. Enantioselective Scheme 1. Enantioselective preparation preparation of of cyclohexanones cyclohexanones (R)-1a (R)-1aand and(R)-1b. (R)-1b. The In asatisfactory similar way, results achieved in the the asymmetric ATH step reduction was prompted performed us to examine with the scope of the enantiomeric the reaction with other cyclohexenones 8–10 using the optimized (S,S)-5 catalyst, providing allylic alcohol (S)-6 in 78% yield, along with traces of the totally conditions ((R,R)-5, TBAC and hydrogenated derivative. Hydrolysis of the ketal delivered (S)-1a in 62% yield anddeliv- HCOONa in CH 2 Cl 2 /H 2 O 1:1, Table 1). The ATH reaction on 2-cyclohexenone 8 92% ered a 2:1 mixture ee (determined byofCHPLC), the corresponding while silylalcohol 11 and the fully ether protection hydrogenated and ketal cleavage cyclohexanol afforded (S)- 1biningood 12 88% yield yield.with Thus,an we enantiomeric establishedexcess of 92%enantioselective a practical (entry 1). The ATH on cyclohexenone procedure to prepare bearing 9both enantiomers of 4-hydroxy-2-cyclohexenone (R)- and (S)-1a in four corresponding a methyl group at the β position, provided a 10:1 mixture of the steps, ca. 44% allylic overallalcohol yield and 13 and92%itsee, fully and hydrogenated their O-silylderivative derivatives, fromand 14,(R)- which(S)-1b,the major in fivecompound steps and 60% overall yield, which compared with other published methodologies [5–13].ofBoth 13 could be isolated by column chromatography (entry 2). The enantioselectivity the reaction was slightly lower (88% ee). Finally, the ATH reaction enantiomers of the product could be obtained from the same precursor only by selecting with 1-tetralone 10 (entry 3) delivered allylic alcohol 15 in good yield with the highest enantioselectivity (94% ee). either the (S,S)- or (R,R)-5 catalyst. Overall, the ATH reaction on 8–10 in the presence of (R,R)-5 proved to be highly effective, The satisfactory results achieved in the ATH step prompted us to examine the scope providing access to the corresponding alcohols as the major products with excellent levels of the reaction with other cyclohexenones 8–10 using the optimized conditions ((R,R)-5, of enantioselectivity. The enantio- and chemoselectivities of the ATH reaction were sensi- TBAC and HCOONa in CH2Cl2/H2O 1:1, Table 1). The ATH reaction on 2-cyclohexenone tive to the structure of the cyclohexenones. In cyclohexanone 8, the formation of the fully 8 delivered a 2:1 mixture of the corresponding alcohol 11 and the fully hydrogenated hydrogenated compound was detrimental for the yield. The presence of a vinylic methyl cyclohexanol 12 in good yield with an enantiomeric excess of 92% (entry 1). The ATH on group enhances the chemoselectivity of the ATH reaction. These chiral cyclohexanols are cyclohexenone 9 bearing a methyl group at the β position, provided a 10:1 mixture of the useful platforms for the synthesis of more elaborate products [26–28]. The ATH reaction corresponding allylic alcohol 13 and its fully hydrogenated derivative 14, from which the has also been studied on cycloalkenones, with five- and seven-membered rings obtaining major compound 13 could be isolated by column chromatography (entry 2). The Int. J. Mol. Int. Int. Int.Int. Int. J.J. Mol. Mol. Sci. J. Mol. J.J. Mol. Mol. 2022, Sci. Sci. Sci.Sci. Sci. 2022, 23, 2022, 2022, x FOR 23, 23, 23,23, 2022, 2022, 23, x FOR xx FOR xx FOR FOR FOR lower PEER REVIEW PEER PEER PEER PEER values of enantio- and chemoselectivity (see the Supplementary REVIEW REVIEW REVIEW PEER REVIEW REVIEW 4 of 44 of 4Materials). 15 of 44 of of of 15 15 15 15 15 enantioselectivity of the reaction was slightly lower (88% ee). Finally, the ATH reaction with 1-tetralone 10 (entry 3) delivered Table 1. Asymmetric Hydrogenation of Cyclic Ketones. allylic alcohol 15 in good yield with the highest enantioselectivity Table Table 1. Table Table Asymmetric 1. Asymmetric 1. Asymmetric Asymmetric 1. Asymmetric Table Table 1. Asymmetric 1. (94% ee). Overall, Hydrogenation Hydrogenation Hydrogenation Hydrogenation Hydrogenation Hydrogenation of of Cyclic of of of of Cyclic Cyclic Cyclic Cyclic the ATH reaction on 8–10 in the presence of (R,R)-5 Ketones. Cyclic Ketones. Ketones. Ketones. Ketones. Ketones. proved to be highly effective, providing access to the corresponding a alcohols as b Entry Entry Entry Substrate Substrate Substrate Products Products Products Yield Yield Yield(%) (%)(%) Ratio Ratio Ratio a aa eeee (%) (%)b bb eethe (%)major Entry Entry Entry Substrate Substrate Substrate Products Products Products Yield Yield Yield (%) (%) Ratio (%) Ratio Ratio eeee aa a ee (%) (%) bb b (%) products with excellent levels of enantioselectivity. The enantio- and chemoselectivities of the ATH reaction were sensitive to the structure ccc cc of the cyclohexenones. In 1111 11 82 82 82 8282 2:12:1 2:1 92 2:12:1 92 92was 92 cyclohexanone 8, the formation of the fully hydrogenated 82 c c compound 2:1 92 92 detrimental for the yield. The presence of a vinylic methyl group enhances the chemoselectivity of the ATH reaction. These chiral cyclohexanols are useful platforms for the synthesis of more elaborate products [26–28]. The ATH reaction has 65 also 65 been studied8888 on cycloalkenones, 2222 22 65 6565 10:1 10:1 10:1 10:188 10:1 88 88 with five- and seven-membered rings obtaining 65 lower10:1 values88of enantio- and chemoselectivity (see the Supplementary Materials). 33 8787 87 - - 9494 333 3 8787 87 -- - 9494 - 94 94 a aa Determined a Determined aa by byby Determined Determined by 1H-NMR. 1H-NMR. 11 b Determined H-NMR. 11H-NMR. b Determined bb by Determined bb Determined chiral byby by high-pressure chiral chiral chiral liquid high-pressure high-pressure high-pressure chromatography liquid liquid liquid (CHPLC). chromatography chromatography chromatography (CHPLC). (CHPLC). (CHPLC). a Determined Determined Determined c From byby by H-NMR.bDetermined H-NMR. 1 H-NMR. Determined Determined byby by chiral chiralchiralhigh-pressure high-pressure high-pressure liquid liquid chromatography chromatography liquid chromatography (CHPLC). (CHPLC). (CHPLC). c From the cc From From cc FromFrom c From the mixture the the thethe mixture mixture mixture of of ofcompounds of of compounds compounds compounds 11 1111and 11 andand and12. 12. 12. 12.12. mixture the of mixture mixture compounds of compounds and 12.11 11 compounds 11 and and 12. Next, Next, Next, we we wewe turned turned turned our ourour attention attention attention to totothe to the use the use of use chiral of ofof chiral chiral cyclohexanol cyclohexanol cyclohexanol R-(6) R-(6) R-(6) as the as asas thethestarting starting starting Next, Next, we turned turned ourour attention attention to thetheuseuse of chiral chiral cyclohexanol cyclohexanol R-(6) R-(6) as thethe starting starting material material material in in the in thethe preparation preparation preparation of of the of thethecytosine cytosine cytosine analogue analogue analogue 2e, 2e, following 2e, following following an ananadaptation adaptation adaptation of of our of ourour material material in in thethe preparation preparation of of thethe cytosine cytosine analogue analogue 2e,2e, following following anan adaptation adaptation of of our our earlier earlierwork earlier work[14]. work [14].Accordingly, [14]. Accordingly,the Accordingly, thethesynthesis synthesisof synthesis of2e ofof 2e(Scheme 2e2e (Scheme2) (Scheme 2)started 2)2) startedwith started withthe with the the earlierwork earlier work[14]. [14].Accordingly, Accordingly,the thesynthesis synthesisof 2e (Scheme2) (Scheme startedwith started withthe the cyclopropanation cyclopropanationreaction cyclopropanation reactionon reaction on on(R)-6, (R)-6,which (R)-6, whichwas which wasfirst was firstattempted first attemptedusing attempted usingShi’s using Shi’scarbenoid Shi’s carbenoid carbenoid cyclopropanation cyclopropanation reaction reaction onon (R)-6, (R)-6, which which waswas first first attempted attempted using using Shi’s Shi’s carbenoid carbenoid (CF (CF(CF 3COOZn 3COOZn COOZn CH CH CH 2I) [29,30]. I) [29,30]. I)222[29,30]. [29,30]. Under Under Under these these these conditions, conditions, conditions, the thethecyclopropanation cyclopropanation cyclopropanation took took took place place place with with with (CF (CF 33COOZn 33COOZn CH CH22I) I) [29,30]. Under Under these these conditions, conditions, thethe cyclopropanation cyclopropanation took took place place with with concomitant concomitant concomitant removal removal removal of ofofthe of thetheketal ketal ketal to totodeliver to deliver deliver the the known the known known bicyclic keto bicyclic bicyclic keto keto alcohol alcohol alcohol 16 albeit 16 1616 albeit albeit in in inin concomitant concomitant removal removal of the the ketal ketal to deliver deliver thethe known known bicyclic bicyclic keto keto alcohol alcohol 16 albeit albeit in Int. J. Mol. Sci. 2022, 23, 9704 4 of 15 Next, we turned our attention to the use of chiral cyclohexanol R-(6) as the starting material in the preparation of the cytosine analogue 2e, following an adaptation of our earlier work [14]. Accordingly, the synthesis of 2e (Scheme 2) started with the cyclopropa- nation reaction on (R)-6, which was first attempted using Shi’s carbenoid (CF3COOZn CH2I) [29,30]. Under these conditions, the cyclopropanation took place with concomi- tant removal of the ketal to deliver the known bicyclic keto alcohol 16 albeit in very low yield (16%). Better results were obtained using Furukawa’s procedure [31,32] (Et2 Zn and ICH2 Cl), which, after purification by column chromatography, afforded 16 in 78% yield. This compound was highly volatile; therefore, the next protection step was performed immediately after the cyclopropanation reaction without further purification. To continue with the synthesis, two protecting groups were evaluated. First, protection of the alcohol as a tert-butyldiphenylsilyl ether furnished 17 in 22% yield for the two steps. All attempts to increase the yield failed. On the other hand, benzyl protection under standard conditions (BnBr, NaH, THF) did not provide better results, delivering only unidentified decomposi- tion products. After some experimentation, it was found that the O-benzyl-protected 18 could be obtained in 62% overall yield over the two steps, using BnBr and Ag2 O. Next, olefination of the ketone via a Wittig reaction led to the expected alkene 19, which was rapidly submitted to the next step, as it easily isomerizes to the endocyclic isomer 20. Accordingly, the subsequent hydroboration (9-BBN) -oxidation (H2 O2 ) process furnished a chromatographically inseparable 2:1 mixture of diastereomers 21 and 22 in 95% yield. After several purifications by column chromatography, an enriched fraction of the main product was obtained and analyzed by NMR. The anti relative configuration of C-20 of the main product was determined by a NOESY experiment that showed cross peaks between H-70 endo and H-20 , indicating that the approach of the borane through the syn face was favored, due to the steric hindrance between the alkylborane and the cyclopropane in the four-center transition state that resulted from the approach of the borane to the more accessible anti face. After purification by column chromatography, benzoyl protection, followed by the removal of the benzyl-protecting group, delivered alcohol 23 in 62% yield and its diastereomer 24 in 29% yield. The primary amine 25 required for the nucleobase construction was accomplished in 84% yield, starting from 23 through a Mitsunobu reaction using diphenylphosphoryl azide (DPPA) [33], followed by catalytic hydrogenation and hydrochloride salt formation. The cytosine nucleoside was prepared through the amination of the corresponding uridine derivative, which was prepared by a two-step protocol that involved the addition of the amine 25 to the isocyanate 26, followed by acid-mediated cyclization, to furnish the protected uracil nucleoside 27 [34]. Then, a one-pot amination in the presence of TsCl, Et3 N and N-methylpiperidine at 0 ◦ C [35], followed by ammonolysis with 30% NH4 OH solution in water and, finally, removal of the benzoyl protection (CH3 NH2 , 33% in EtOH), afforded, after purification by column chromatography, the desired cytosine nucleoside analogue 2e. Compound 2e was examined for antiherpetic activity (herpes simplex virus-1 (HSV-1; strain KOS) and herpes simplex virus-2 (strain G) in human embryonic lung (HEL) cell cultures. Unfortunately, however, it did not show significant antiviral activity at subtoxic concentrations (~100 µM) (see Supplementary Materials). These results were similar to those obtained for the previously synthesized compounds 2a–d. As previously mentioned, the lack of significant activity against herpes simplex virus by the 50 -hydroxymethylbicyclo [4.1.0]heptanyl NAs may arise from a low affinity (if any) or from substrate activity for the viral-encoded or cellular nucleoside kinases. Therefore, to evaluate this last point, we performed further studies to test the suitability of these compounds to pass the usual rate-limiting first phosphorylation step. Thus, the affinity of the synthesized compounds 2a–e for the cellular and HSV-1 thymidine kinases (TKs) were investigated (Table 2). Based on these results, it can be concluded that compounds 2b and, to a lesser extent, 2d, were nicely recognized by herpes simplex virus-TK (IC50 = 1.6 ± 0.1 µg/mL and 52 ± 28 µg/mL, respectively), in agreement with our modelling studies [14]. Only compound 2b showed some marginal affinity for mitochondrial TK-2, in addition to its excellent affinity for HSV-1 Int. J. Mol. Sci. 2022, 23, 9704 5 of 15 Int. J. Mol. Sci. 2022, 23, x FOR PEERTK. REVIEW The 2b analogue was also evaluated for substrate activity by HPLC technology, and 5 it was found to be a good substrate for HSV-1 TK with an efficient conversion to the monophosphate metabolite. The cytosine derivative 2e did not display affinity for HSV-1 TK. It was expected that this cytosine analogue might show affinity for HSV-1 TK, as the of the nucleoside natural amine 25 to the isocyanate2-dC) (2-deoxycytidine, 26, followed is known to bybe acid-mediated cyclization, recognized by several to furnis kinases, protected including uracil HSV-1 TK;nucleoside however, that 27was[34]. Then, anot apparently one-pot the case.amination in none Interestingly, the presence of the of Et3N andshowed analogues N-methylpiperidine significant affinityatto0human °C [35], followed cellular byTherefore, kinases. ammonolysis becausewith some30% NH of the synthesized solution in wateranalogues wereremoval and, finally, recognized of by thethe virus-encoded benzoyl nucleoside protection (CH3NH kinase, 2, 33% in Et the lack of antiherpetic activity could be attributed to the lack of further conversion of afforded, after purification by column chromatography, the desired cytosine nucleo the 50 -monophosphate derivative to the 50 -triphosphate or to a low affinity, if any, of the analogue 2e. 50 -triphosphate metabolite to the virus-encoded DNA polymerase. Scheme Scheme 2. 2. Synthesis Synthesis of compound of compound 2e. 2e. Compound 2e was examined for antiherpetic activity (herpes simplex virus-1 (H 1; strain KOS) and herpes simplex virus-2 (strain G) in human embryonic lung (HEL cultures. Unfortunately, however, it did not show significant antiviral activity at sub concentrations (~100 μM) (see Supplementary Materials). These results were simil Int. J. Mol. Sci. 2022, 23, 9704 6 of 15 Table 2. Evaluation of the synthesized compounds for affinity against nucleoside kinases. IC50 a Compound Conc. Unit Human TK-1 Human TK-2 HSV-1 TK 2a µg/mL >200 >200 150 ± 15 2b µg/mL >200 93 ± 7 1.6 ± 0.1 2c µg/mL >200 >200 >200 2d µg/mL >200 >200 52 ± 28 2e µg/mL >100 >100 >100 a 50% inhibitory concentration, or compound concentration required to inhibit TK-catalyzed conversion of [3H]dThd to its 50 -monophosphate derivative. 3. Materials and Methods General Methods. Commercially available reagents were used as received. Solvents were dried by distillation over the appropriate drying agents. All of the reactions were monitored by analytical thin-layer chromatography (TLC), using silica gel 60 F254 pre- coated aluminum plates (0.25 mm thickness). TLC spots were detected under UV light and/or by charring with a KMnO4 /KOH aqueous solution or vanillin solution. Flash column chromatography was performed using silica gel (230–400 mesh). 1 H NMR spectra were recorded using 250 or 400 MHz and were referenced to the residual proton signals of CDCl3 , 7.26 ppm, and MeOH-d4 , 3.31 ppm. 13 C [1 H] NMR spectra were recorded at 90 MHz or 100 MHz and were referenced to the residual 13 C signal of CDCl3 , 77.16 ppm, and MeOH-d4 , 49.00 ppm. NMR signals were assigned with the help of COSY, HSQC, HMBC, and NOESY experiments. Melting points were determined on a hot stage and were uncorrected. Optical rotations were measured at 20 ± 2 ◦ C at the sodium D line (589 nm) in a microcell (0.1 dm). Infrared spectra were recorded on a spectrophotometer equipped with a Golden Gate Single Refraction Diamond ATR (attenuated total reflectance) accessory. High-resolution mass spectra were recorded using electrospray ionization (ESI). Thymidine Kinase Assay Using [CH3 -3 H]dThd as the Natural Substrate [36]. The activity of recombinant thymidine kinase 1 (TK-1), TK-2, and herpes simplex virus-1 (HSV- 1) TK, and the 50% inhibitory concentration of the test compounds, were assayed in a 50-µL reaction mixture containing 50 mM Tris/HCl, pH 8.0, 2.5 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM CHAPS, 3 mg/mL bovine serum albumin, 2.5 mM ATP, 1 µM [methyl-3 H]dThd, and enzyme. The samples were incubated at 37 ◦ C for 30 min in the presence or absence of different concentrations (five-fold dilutions) of the test compounds. At this time, the enzyme reaction still proceeded linearly. Aliquots of 45 µL of the reaction mixtures were spotted on Whatman DE-81 filter paper disks (Whatman, Clifton, NJ, USA). The filters were washed three times for 5 min each in 1 mM ammonium formate, once for 1 min in water, and once for 5 min in ethanol. The radioactivity was determined by scintillation counting. Synthetic Procedures (8R)-1,4-Dioxaspiro [4.5]dec-6-en-8-ol [(R)-6]. To a stirred solution of enone 4 (104 mg, 0.68 mmol) in a biphasic medium of CH2 Cl2 (2.3 mL) and water (2.3 mL), tetrabutylammo- nium chloride (57 mg, 0.20 mmol), sodium formate (139 mg, 2.04 mmol), and (R,R)-Noyori-I [(R,R)-5] catalyst (13 mg, 0.020 mmol) were sequentially added at room temperature. The reaction mixture was allowed to stir for 3 h. Then, the phases were separated, and the aqueous layer was extracted with additional CH2 Cl2 (2 × 2 mL) and the organic layers were concentrated under reduced pressure, affording a yellow oil that was purified by column chromatography (diethyl ether) to furnish allylic alcohol (R)-6 (83 mg, 0.53 mmol, 79%): [α]D 20 = +39.0 (c 1.2, CHCl3 ) [lit: [37] [α]D 20 = +39.8 (c 1.21, CHCl3 ), ee = 93.1%] as a yellowish oil: 1 H NMR (250 MHz, CDCl3 ) δ 5.94 (ddd, J7,6 = 10.1 Hz, J7,8 = 2.8 Hz, J7,9 = 1.1 Hz, 1H, H-7), 5.61 (dt, J6,7 = 10.1 Hz, J6,8 = J6,10 =1.5 Hz, 1H, H-6), 4.29–4.12 (m, 1H, H-8), 4.05–3.85 (m, 4H, H-2, H-3), 2.17–2.02 (m, 1H, H-9), 2.00–1.89 (m, 1H, H-9), 1.85–1.65 (m, 2H, H-10); 13 C NMR (90 MHz, CDCl3 ) δ 135.3 (C-7), 129.0 (C-6), 105.2 (C-5), 66.0 (C-8), 64.8/64.6 (C-2, C-3), 31.0/30.6 (C-9, C-10). Int. J. Mol. Sci. 2022, 23, 9704 7 of 15 (8S)-1,4-Dioxaspiro [4.5]dec-6-en-8-ol [(S)-6]. To a stirred solution of enone 4 (76 mg, 0.49 mmol) in a biphasic medium of CH2 Cl2 (1.3 mL) and water (1.3 mL), tetrabutylammo- nium chloride (45 mg, 0.16 mmol), sodium formate (140 mg, 2.06 mmol), and (S,S)-Noyori-I catalyst, [(S,S)-5], (9.1 mg, 0.015 mmol) were sequentially added at room temperature. The reaction mixture was allowed to stir for 24 h. Then, the phases were separated, and the aque- ous layer was extracted with CH2 Cl2 (2 × 10 mL). The organic layers were concentrated under reduced pressure, affording a yellow oil that was purified by column chromatogra- phy (diethyl ether) to obtain allylic alcohol (S)-6 (60 mg, 0.38 mmol, 78% yield) as a yellow oil: [α]D 20 = −38.7 (c 1.3, CHCl3 ) [lit: [38] [α]D 20 = −40.5 (c 1.24, CHCl3 ) ee > 98.4%]; 1 H NMR (250 MHz, CDCl3 ) δ: 5.93 (ddd, J7,6 = 10.1 Hz, J7,8 = 2.8 Hz, J7,9 = 1.0 Hz, 1H, H-7), 5.60 (dt, J6,7 = 10.1 Hz, J6,8 = J6,10 = 1.4 Hz, 1H, H-6), 4.26–4.16 (m, 1H, H-8), 4.05–3.85 (m, 4H, H-2, H-3), 2.19–2.05 (m, 1H, H-9), 2.04–1.90 (m, 1H, H-9), 1.90–1.68 (m, 2H, H-10). (4R)-4-Hydroxycyclohex-2-en-1-one [(R)-1a]. Montmorillonite K-10 (832 mg) was added to a solution of (R)-6 (77 mg, 0.49 mmol) in CH2 Cl2 (7.6 mL) and the mixture was stirred at room temperature for 4 h. Then, it was filtered, and the solvent was removed under vacuum to furnish the enone (R)-1a (34 mg, 0.31 mmol, 64% yield) as a yellowish oil: CHPLC (Daicel Chiralpak IC): 92% ee; [α]D 20 = +90.0 (c 0.2, CHCl3 ) [lit: [39] [α]D 20 = +92.3 (c 0.7, CHCl3 ) ee > 99%]; 1 H NMR (250 MHz, CDCl3 ) δ 6.95 (ddd, J3,2 = 10.2 Hz, J3,4 = 2.1 Hz, J3,5eq = 1.6 Hz, 1H, H-3), 5.98 (ddd, J2,3 = 10.2 Hz, J2,4 = 2.1 Hz, J2,6 = 1.0 Hz, 1H, H-2), 4.58 (ddt, J4,5ax = 9.2 Hz, J4,5eq = 4.4 Hz, J4,2 =J4,3 =2.1 Hz, 1H, H-4), 2.60 (dt, Jgem = 9.8 Hz, J6eq,5 = J6eq,4 = 4.5 Hz, 1H, H-6eq), 2.45–2.28 (m, 2H, H-6ax, H-5eq), 2.10–1.90 (m, 1H, H-5ax); 13 C NMR (90 MHz, CDCl3 ) δ 199.4 (C-1), 153.6 (C-3), 129.0 (C-2), 66.2 (C-4), 35.4 (C-6), 32.4 (C-5). (4S)-4-Hydroxycyclohex-2-en-1-one [(S)-1a]. Montmorillonite K-10 (852 mg) was added to a solution of (S)-6 (79 mg, 0.50 mmol) in CH2 Cl2 (7.8 mL) and the mixture was stirred at rt for 4 h. Then, it was filtered and the solvent was removed under vacuum to furnish the known enone (S)-1a (35 mg, 0.31 mmol, 62% yield) as a yellowish oil: CHPLC (Daicel Chiralpak IC): 92% ee; [α]D 20 =−92.3 (c 0.50, CHCl3 ) [lit: [9] [α]D 20 = −92.0 (c 0.50, CHCl3 ) ee > 99%]; 1 H NMR (250 MHz, CDCl3 ) δ 6.93 (ddd, J3,2 = 10.2 Hz, J3,4 = 2.4 Hz, J3,5eq = 1.6 Hz, 1H, H-3), 5.95 (ddd, J2,3 = 10.2 Hz, J2,4 = 2.0 Hz, J2,6 = 1.0 Hz, 1H, H-2), 4.68–4.46 (m, 1H, H-4), 2.80–2.68 (m, 1H, OH), 2.57 (ddd, Jgem = 9.8 Hz, J6eq,4 = 5.0Hz, J6eq,5 = 4.0 Hz, 1H, H-6eq), 2.47–2.24 (m, 2H, H-6ax, H-5eq), 2.00 (ddd, Jgem = 12.8 Hz, J5ax,6ax = 9.5 Hz, J5ax,4 = 5.3 Hz, 1H, H-5ax). (4R)-4-((tert-Butyldimethylsilyl)oxy)cyclohex-2-en-1-one [(R)-1b]. To a stirred solu- tion of (R)-6 (102 mg, 0.65 mmol) in CH2 Cl2 (0.5 mL), imidazole (56 mg, 0.82 mmol) and a solution of TBSCl (124 mg, 0.82 mmol) in CH2 Cl2 (0.4 mL) were added at room temper- ature. The mixture was allowed to stir overnight. Then, a saturated aqueous NaHCO3 solution (1 mL) was added, the aqueous layer was extracted with CH2 Cl2 (3 × 1 mL), and the organic layers were dried and concentrated under vacuum. The crude was dis- solved in CH2 Cl2 (4 mL) and montmorillonite K-10 (513 mg) was added. The reaction mixture was allowed to stir at room temperature for 1 h. Then, it was filtered, and the solvent was removed under vacuum to furnish enone (R)-1b (128 mg, 0.56 mmol, 87% yield) as a yellowish oil: [α]D 20 = +92.3 (c 1.02, CHCl3 ) [lit: [39] [α]D 20 = +97.9 (c 1.2, CHCl3 ) ee > 99%]; 1 H NMR (400 MHz, CDCl3 ) δ 6.82 (ddd, J3,2 = 10.2 Hz, J3,4 = 2.1 Hz, J3,5eq = 1.6 Hz, 1H, H-3), 5.91 (ddd, J2,3 = 10.2 Hz, J2,4 = 2.1 Hz, J2,6eq = 1.0 Hz, 1H, H-2), 4.51 (ddt, J4,5ax = 9.1 Hz, J4,5eq = 4.6 Hz, J4,2 = J4,3 = 2.1 Hz, 1H, H-4), 2.56 (dt, Jgem = 16.8 Hz, J6eq,5ax = J6eq,5eq = 4.5 Hz, 1H, H-6eq), 2.33 (ddd, Jgem = 16.8 Hz, J6ax,5ax = 12.8 Hz, J6ax,5eq = 4.7 Hz, 1H, H-6ax), 2.20 (dqd, Jgem = 12.8 Hz, J5eq,4 = J5eq,6eq = J5eq,6ax = 4.6 Hz, J5eq,3 = 1.6 Hz, 1H, H-5eq), 1.99 (tdd, Jgem = J5ax,6ax = 12.8 Hz, J5ax,4 = 9.1 Hz, J5ax,6eq = 4.5 Hz, 1H, H-5ax), 0.90 (s, 9H, H-tBu), 0.12 (s, 3H, SiCH3 ), (s, 3H, SiCH3 ). (4S)-4-((tert-Butyldimethylsilyl)oxy)cyclohex-2-en-1-one [(S)-1b]. To a stirred solu- tion of (S)-6 (49 mg, 0.32 mmol) in CH2 Cl2 (0.4 mL), imidazole (34 mg, 0.49 mmol) and a solution of TBSCl (74 mg, 0.49 mmol) in CH2 Cl2 (0.2 mL) were added at room temperature. The mixture was allowed to stir overnight. Then, a saturated aqueous NaHCO3 solution Int. J. Mol. Sci. 2022, 23, 9704 8 of 15 (1 mL) was added, the aqueous layer was extracted with more CH2 Cl2 (3 × 1 mL), and the organic layers were dried and concentrated under vacuum. The crude was dissolved in CH2 Cl2 (4 mL) and montmorillonite K-10 (294 mg) was added. The reaction mixture was allowed to stir at room temperature for 1 h. Then, it was filtered, and the solvent was removed under reduced pressure to afford enone (S)-1b (64 mg, 0.28 mmol, 88% yield) as a yellowish oil: [α]D 20 = −93.7 (c 0.70, CHCl3 ) [lit: [6] [α]D 20 = −97.0 (c 1.26, CHCl3 ), ee >99%]; 1 H NMR (250 MHz, CDCl ) δ 6.81 (ddd, J 3 3,2 = 10.2 Hz, J 3,4 = 2.1 Hz, J 3,5eq = 1.6 Hz, 1H, H-3), 5.90 (ddd, J2,3 = 10.2 Hz, J2,4 = 2.1 Hz, J2,6eq = 1.0 Hz, 1H, H-2), 4.50 (ddt, J4,5ax = 9.0 Hz, J4,5eq = 4.8 Hz, J4,3 = J4,2 = 2.1 Hz, 1H, H-4), 2.55 (dt, Jgem = 16.7 Hz, J6eq.5ax = J6eq,5eq = 4.8 Hz, 1H, H-6eq), 2.32 (ddd, Jgem = 16.7 Hz, J6ax,5ax = 12.8 Hz, J6ax,5eq = 4.8 Hz, 1H, H-6ax), 2.19 (dqd, Jgem = 12.8 Hz, J5eq,6eq = J5eq,4 = J5eq,6ax = 4.8 Hz, J5eq,3 = 1.6 Hz, 1H, H-5eq), 1.97 (tdd, Jgem = J5ax,6ax = 12.8 Hz, J5ax,4 = 9.0 Hz, J5ax,6eq = 4.8 Hz, 1H, H-5ax), 0.89 (s, 9H, H-tBu), 0.11 (s, 3H, SiCH3 ), 0.10 (s, 3H, SiCH3 ). (1R)-2-Cyclohexen-1-ol [(R)-11]. To a stirred solution of enone 8 (250 µL, 2.59 mmol) in a biphasic medium of CH2 Cl2 (8.6 mL) and water (8.6 mL) and under a nitrogen flux, tetrabutylammonium chloride (222 mg, 0.8 mmol), sodium formate (532 mg, 7.82 mmol), and (R,R)-Noyori-I catalyst, [(R,R)-5] (49.6 mg, 0.08 mmol) were sequentially added at room temperature. The reaction mixture was allowed to stir for 24 h. Then, CH2 Cl2 (10 mL) and water (10 mL) were added, the two phases were separated, and the aqueous layer was extracted with CH2 Cl2 (3 × 5 mL). The volatiles of the organic phase were removed under reduced pressure and the resulting residue was purified by column chromatography (CHCl3 ), affording an inseparable mixture of allylic alcohol (R)-11 and cyclohexanol 12 (210 mg, 2.14 mmol, 82% yield) in a 2:1 ratio as a yellowish oil that was analyzed by NMR spectroscopy and CHPLC (Daicel Chiralpak IC): 92% ee; 1 H NMR (250 MHz, CDCl3 ) ((R)-11) δ 5.82 (dtd, J3,2 = 10.0 Hz, J3,4eq = J3,4ax = 3.3 Hz, J3,1 = 1.1 Hz, 1H, H-3), 5.73 (ddt, J2,3 = 10.0, J2,1 = 3.4, J2,4 = J2,6 = 1.8 Hz, 1H, H-2), 4.42–4.13 (m, 1H, H-1), 2.07–1.89 (m, 1H, H-6), 1.92–1.81 (m, 1H, H-6), 1.79–1.64 (m, 2H, H-4, H-5), 1.64–1.48 (m, 2H, H-4, H-5); (12) δ 3.66–3.52 (m, 1H, H-10 ), 1.64–1.48 (m, 4H, H-20 , H-60 ), 1.38–0.96 (m, 6H, H-30 , H-40 , H-50 ). (1R)-3-Methyl-2-cyclohexen-1-ol [(R)-13]. To a stirred solution of enone 9 (510 µL, 495 mg, 4.49 mmol) in a biphasic medium of CH2 Cl2 (14.7 mL) and water (14.7mL) and under a nitrogen flux, tetrabutylammonium chloride (367 mg, 1.32 mmol), sodium formate (899 mg, 13.2 mmol), and (R,R)-Noyori-I catalyst [(R,R)-5] (84 mg, 0.13 mmol) were sequen- tially added at room temperature. The reaction mixture was allowed to stir for 72 h. Then, CH2 Cl2 (10 mL) and water (10 mL) were added, the two phases were separated, and the aqueous layer was extracted with CH2 Cl2 (3 × 5 mL). The volatiles of the organic phase were removed under reduced pressure and the resulting residue was purified by column chromatography (CHCl3 ), yielding allylic alcohol (R)-13 (327 mg, 2.92 mmol, 65% yield) as a yellowish oil: CHPLC (Daicel Chiralpak IC): 88% ee; [α]D 20 = +89.8 (c 0.1, CHCl3 ) [lit: [40] [α]D 20 = +62.4 (c 1.0, CHCl3 ), ee = 96%]; 1 H NMR (250 MHz, CDCl3 ) δ 5.47 (dq, J2,1 = 3.3 Hz, J2,10 = 1.6 Hz, 1H, H-2), 4.22–4.02 (m, 1H, H-1), 2.06–1.81 (m, 2H, H-6), 1.86–1.57 (m, 2H, H-5), 1.67 (s, 3H, H-10 ), 1.64–1.34 (m, 2H, H-4). (1R)-1,2,3,4-Tetrahydro-1-naphthalenol [(R)-15]. To a stirred solution of α-tetralone 10 (100 µL, 110 mg, 0.73 mmol) in a biphasic medium of CH2 Cl2 (2.4 mL) and water (2.4 mL) and under a nitrogen flux, tetrabutylammonium chloride (61 mg, 0.22 mmol), sodium formate (149 mg, 2.19 mmol), and (R,R)-Noyori-I catalyst [(R,R)-5] (13.9 mg, 0.022 mmol) were sequentially added at room temperature. The reaction mixture was allowed to stir for 24 h. Then, CH2 Cl2 (2 mL) and water (2 mL) were added, the two phases were separated, and the aqueous layer was extracted with CH2 Cl2 (3 × 1 mL). The volatiles of the organic phase were removed under reduced pressure and the resulting residue was purified by column chromatography (CHCl3 ), yielding alcohol (R)-15 (97 mg, 0.65 mmol, 87% yield) as a yellowish oil: CHPLC (Daicel Chiralpack OD-H): 94% ee; [α]D 20 = −36.5 (c 0.98, CHCl3 ) [lit: [40] [α]D 20 = −33.2 (c 1.0, CHCl3 ), ee = 99%]; 1 H NMR (250 MHz, CDCl3 ) δ 7.43 (dd, J8,7 = 5.5 Hz, J8,6 = 3.5 Hz, 1H, H-8), 7.25–7.17 (m, 2H, H-6, H-7), 7.11 (dd, J5,6 = 5.5 Hz, J5,7 = 3.5 Hz 1H, H-5), 4.76 (t, J1,2ax = J1,2eq = 4.5 Hz, 1H, H-1), 2.94–2.62 (m, 2H, H-4), Int. J. Mol. Sci. 2022, 23, 9704 9 of 15 2.05–1.84 (m, 4H, H-2, H-3); 13 C NMR (100 MHz, CDCl3 ) δ 138.9 (C-8a), 137.3 (C-4a), 129.2/128.8 (C-5, C-8), 127.7/126.3 (C-6, C-7), 68.3 (C-1), 32.4 (C-2), 29.4 (C-4), 18.9 (C-3). (1R,5R,6S)-5-(Benzyloxy)bicyclo [4.1.0]heptan-2-one (18). To a stirred solution of allylic alcohol (R)-6 (314 mg, 2.01 mmol) in anhydrous CH2 Cl2 (10 mL), Et2 Zn (4 mL, 4.02 mmol, 1 M in hexane) at 0 ◦ C was added and the mixture was stirred for 5 min at this temperature. Then, a solution of ICH2 Cl (600 µL, 8.30 mmol) in CH2 Cl2 (3 mL) was added dropwise via syringe at 0 ◦ C, and the mixture was stirred overnight, allowing it to warm to room temperature. Then, a saturated aqueous NaHCO3 solution (10 mL) was added, and the aqueous layer was extracted with CH2 Cl2 (3 × 10 mL). The organic layer was dried (Na2 SO4 ), concentrated under reduced pressure (with vacuum controller) and purified by column chromatography (hexane-EtO2 , 4:1) furnishing ketone 16 (198 mg, 1.57 mmol, 78% yield) as a colorless oil. Due to the volatility of compound 16, the crude was used without further purification prior to the next step. To a solution of crude of the hydroxyacetone 16 in CH2 Cl2 (4 mL), Ag2 O (560 mg, 2.41 mmol) and benzyl bromide (340 µL, 2.81 mmol) were added. The mixture was stirred at room temperature for 24 h, then it was filtered through a Celite® pad and concentrated in vacuo. The crude was purified by column chromatography (hexanes-EtOAc, 6:1) to furnish 18 (270 mg, 1,24 mmol, 62% yield from (R)-6) as a pale oil. 16: [α]D 20 = +78.5 (c 0.65, CHCl3 ) [lit: [41] [α]D 20 = −80.7 (c 1.37, CHCl3 ), for its enan- tiomer, ee = 100%]; 1 H NMR (400 MHz, CDCl3 ) δ 4.42 (dddd, J5,4ax = 10.1 Hz, J5,6 = 5.2 Hz, J5,4eq = 4.1 Hz, J5,3ax = 0.8 Hz, 1H, H-5), 2.37 (ddd, Jgem = 18.4 Hz, J3eq,4ax = 5.6 Hz, J3eq,4eq = 3.6 Hz, 1H, H-3eq), 2.15 (dddd, Jgem = 18.4 Hz, J3ax,4ax = 12.1 Hz, J3ax,4eq = 6.6 Hz, J3ax,5 = 0.8 Hz, 1H, H-3ax), 1.98–1.83 (m, 3H, H-1, H-6, H-4eq), 1.63 (dddd, Jgem = 13.8 Hz, J4ax,3ax = 12.1 Hz, J4ax,5 = 10.3 Hz, J4ax,3eq = 5.6 Hz, 1H, H-4ax), 1.44 (q, Jgem = J7endo,1 = J7endo,6 = 5.4 Hz, 1H, H-7endo), 1.14 (ddd, J7exo,1/6 = 9.8 Hz, J7exo,1/6 = 7.7 Hz, Jgem = 5.4 Hz, 1H, H-7exo); 13 C NMR (100 MHz, CDCl3 ) δ 207.5 (C-2), 65.3 (C-5), 34.8 (C-3), 26.8 (C-1), 26.7 (C-4), 23.3 (C-6), 8.0 (C-7); IR (ATR) ν 3359, 2919, 2850, 1659, 1345, 1066, 1041 (cm−1 ). HRMS (ESI+) Calcd. for [C7 H10 O2 +Na]+ 149.0573, found: 149.0576. 18: [α]D 20 = +62.6 (c 1.45, CHCl3 ); 1 H NMR (400 MHz, CDCl3 ) δ 7.39–7.34 (m, 4H, H-Ar), 7.32–7.28 (m, 1H, H-Ar), 4.73 (d, Jgem = 11.9 Hz, 1H, CH2 -Ph), 4.64 (d, Jgem = 11.9 Hz, 1H, CH2 -Ph), 4.15 (dt, J5,4ax = 9.3 Hz, J5,4eq = J5,6 = 4.9 Hz, 1H, H-5), 2.42 (dt, Jgem = 17.7 Hz, J3eq,4 = 5.5 Hz, 1H, H-3eq), 2.12 (ddd, Jgem = 17.7 Hz, J3ax,4x = 10.7 Hz, J3ax,4eq = 6.5 Hz, 1H, H-3ax), 2.05–1.93 (m, 2H, H-4), 1.88–1.71 (m, 2H, H-1, H-6), 1.50 (q, Jgem = J7endo,6 = J7endo,1 = 5.4 Hz, 1H, H-7endo), 1.24 (td, J7exo,6 = J7exo,1 = 9.1 Hz, Jgem = 5.4 Hz, 1H, H-7exo); 13 C NMR (100 MHz, CDCl3 ) δ 208.2 (C-2), 138.5 (C-Ar), 128.6/128.2/127.9/127.8 (C-Ar), 71.3 (C-5), 70.6 (CH2 -Ph), 34.4 (C-3), 26.5 (C-4), 25.5 (C-1), 21.3 (C-6), 9.5 (C-7); IR (ATR) ν 3028, 2857, 1692 (C=O), 1342, 1075, 1028, 881, 631 cm−1 . HRMS (ESI+) Calcd. for [C14 H16 O2 +H]+ 217.1229, Found: 217.1231. [(1S’,2R’,5R’,6S’)-50 -(Benzyloxy)bicyclo [4.1.0]hept-20 -yl]methanol (21) and its (20 S)- diastereoisomer (22). To a stirring solution of Ph3 PCH3 I (1.452 g, 3.59 mmol) in anhydrous THF (4 mL) at 0 ◦ C, t-BuOK (402 mg, 3.58 mmol) was added, under nitrogen atmosphere, and the resulting yellow mixture was allowed to react for 1 h. Then, a solution of ketone 18 (155 mg, 0.71 mmol) in dry THF (1 mL) was added and the mixture was allowed to warm to room temperature and stirred for 3 h. Then, diethyl ether (10 mL) was added and the crude was filtered through a short pad of silica and Celite® , using additional diethyl ether as eluent. The volatiles were removed under vacuum to obtain an orange oil of alkene 19 that was used for the next step without further purification, as it isomerizes to the more stable endocyclic regioisomer 20 at room temperature. The crude of alkene 19 was rapidly dissolved in anhydrous THF (7 mL) and 9-BBN (4.30 mL, 2.15 mmol, 0.5 M in THF) was added at −10 ◦ C. The mixture was allowed to warm to room temperature and stirred overnight. Then, water (1.2 mL), NaOH (1.5 mL, 3 M in water), and H2 O2 (1.4 mL, 30% in water) were added at 0 ◦ C. After stirring for 15 min, the mixture was diluted with brine (15 mL) and CH2 Cl2 (15 mL) and the aqueous phase was extracted with additional CH2 Cl2 (2 × 10 mL). The organic layers were dried (Na2 SO4 ), Int. J. Mol. Sci. 2022, 23, 9704 10 of 15 concentrated under reduced pressure, and purified by column chromatography (gradient hexane-EtOAc, 5:1 → 2:1) to provide a chromatographically inseparable mixture of alcohols 21 and 22 (158 mg, 0.68 mmol, 95% overall yield from 18) in a ca. 2:1 diastereomeric ratio as a colorless oil. After repeated purification by column chromatography, enriched fractions were obtained and were analyzed by NMR. 19: 1 H NMR (400 MHz, CDCl3 ) δ 7.46–7.25 (m, 5H, H-Ar), 4.90 (br s, 1H, H-1’), 4.79 (br s, 1H, H-10 ), 4.74 (d, Jgem = 11.9 Hz, 1H, CH2 -Ph), 4.56 (d, Jgem = 11.9 Hz, 1H, CH2 -Ph), 4.05 (q, J2,3ax = J2,3eq = J2,1 = 5.9 Hz, 1H, H-2), 2.21 (dddt, Jgem = 14.9 Hz, J4ax,3ax = 8.5 Hz, J4ax,3eq = 4.4 Hz, J4ax,10 = 1.4 Hz, 1H, H-4ax), 2.03 (dddt, Jgem = 14.9 Hz, J4eq,3ax = 7.2 Hz, J4eq,3eq = 4.3 Hz, J4eq,10 = 1.4 Hz, 1H, H-4eq), 1.88–1.75 (m, 1H, H-6), 1.71–1.50 (m, 3H, H-1, H-3), 0.94–0.84 (m, 2H, H-7); 13 C NMR (100 MHz, CDCl3 ) δ 146.3 (C-5), 139.1 (C-ipso), 128.4/127.7/127.5 (C-Ar), 108.1 (C-10 ), 72.0 (C-2), 69.7 (CH2 -Ph), 28.8 (C-4), 27.7 (C-3), 19.9 (C-1), 17.0 (C-6), 8.7 (C-7); IR (ATR) ν 3070, 2930, 1471, 1427, 1106, 806, 741 cm−1 . HRMS (ESI+) Calcd. for [C15 H18 O+H]+ 215.1436, Found: 215.1465. 20: 1 H NMR (400 MHz, CDCl3 ) δ 7.42–7.36 (m, 3H, H-Ar), 7.35–7.25 (m, 2H, H-Ar), 5.01 (dq, J3,4eq = 5.3 Hz, J3,4ax = J3,10 = 1.6 Hz, 1H, H-3), 4.69 (d, Jgem = 12.0 Hz, 1H, CH2 -Ph), 4.65 (d, Jgem = 12.0 Hz, 1H, CH2 -Ph), 3.98 (ddd, J5,4ax = 9.1 Hz, J5,4eq = 6.8 Hz, J5,6 = 4.1 Hz, 1H, H-5), 2.32 (ddd, Jgem = 15.3 Hz, J4eq,5 = 6.8 Hz, J4eq,3 = 5.2 Hz, 1H, H-4eq), 1.89–1.75 (m, 4H, H-10 , H-4ax) 1.64–1.51 (m, 1H, H-6), 1.30–1.23 (m, 1H, H-1), 0.90–0.85 (m, 2H, H-7); 13 C NMR (100 MHz, CDCl3 ): δ 139.6 (C-ipso), 136.7 (C-2), 128.8/128.2/127.9 (C-Ar), 114.3 (C-3), 73.4 (C-5), 70.6 (CH2 -Ph), 28.0 (C-4), 23.5 (C-10 ), 18.0/17.8 (C-1, C-6), 10.1 (C-7); IR (ATR) ν 3064, 2910, 2852, 1667, 1452, 1070, 734, 697 cm−1 . HRMS (ESI+) Calcd. for [C15 H18 O+H]+ 215.1436, Found: 215.1434. 21 and 22 mixture: 1 H NMR (400 MHz, CDCl3 ) (ca. 84% trans-isomer 21) δ 7.40–7.31 (m, 4H, H-Ar), 7.30–7.24 (m, 1H, H-Ar), 4.71 (d, Jgem = 12.0 Hz, 1H, CH2 -Ph), 4.59 (d, Jgem = 12.0 Hz, 1H, CH2 -Ph), 3.95 (dt, J50 ,40 ax = 10.0 Hz, J50 ,40 eq = J50 ,60 = 5.7 Hz, 1H, H- 50 ), 3.57 (d, J1,20 = 6.6 Hz, 2H, H-1), 1.79 (dtd, Jgem = 13.3 Hz, J40 eq,50 = J40 eq,30 ax = 5.7 Hz, J40 eq,30 eq = 2.4 Hz, 1H, H-40 eq), 1.70 (dddd, J20 ,30 ax = 8.5 Hz, J20 ,1 = 6.6 Hz, J20 ,30 eq = 5.4 Hz, J20 ,10 = 1.9 Hz, 1H, H-20 ), 1.55 (dtd, Jgem = 13.2 Hz, J30 eq,40 ax = J30 eq,20 = 5.4 Hz, J30 eq,40 eq = 2.4 Hz, 1H, H-30 eq), 1.29–1.26 (m, 1H, H-60 ), 1.04 (tdd, Jgem = J40 ax,30 ax = 13.3 Hz, J40 ax,50 = 10.0 Hz, J40 ax,30 eq = 5.4 Hz, 1H, H-40 ax), 0.99–0.83 (m, 2H, H-10 , H-30 ax), 0.74 (td, J70 exo,60 = J70 exo,10 = 8.8 Hz, Jgem = 5.4 Hz, 1H, H-70 exo), 0.46 (q, Jgem = J70 endo,60 = J70 endo,10 = 5.4 Hz, 1H, H- 70 endo); 1 H NMR (400 MHz, CDCl3 ) (ca. 16% cis-isomer 22, observable signals) δ 7.40–7.31 (m, 4H, H-Ar), 7.30–7.24 (m, 1H, H-Ar), 4.72 (d, Jgem = 11.8 Hz, 1H, CH2 -Ph), 4.47 (d, Jgem = 11.8 Hz, 1H, CH2 -Ph), 4.01 (ddd, J50 ,40 ax = 7.0 Hz, J50 ,40 eq = 5.1 Hz, J50 ,60 = 3.4 Hz, 1H, H-50 ), 3.57 (m, 1H, H-1a), 3.49 (dd, Jgem = 10.5 Hz, J1b,20 = 6.6 Hz, 1H, H-1b), 2.05 (dddd, J20 ,30 ax = 12.3 Hz, J20 ,1a = 10.2 Hz, J20 ,1b = 6.6 Hz, J20 ,10 = 5.4 Hz, 1H, H-20 ), 1.66–1.57(m, 1H, H-40 a), 1.44–1.33 (m, 2H, H-30 a, H-40 b), 1.18 (tt, J10 ,60 = J10 ,70 exo = 8.5 Hz, J10 ,70 endo = J10 ,20 = 5.4 Hz, 1H, H-10 ), 1.02–0.98 (m, 1H, H-30 b), 0.75 (m, 1H, H-60 ), 0.60–0.51 (m, 2H, H-70 ); 13 C NMR (101 MHz, CDCl ) (ca. 84% trans-isomer 20): δ 139.25 (C-ipso), 128.43 (C-meta), 3 127.82 (C-para), 127.49 (C-orto), 74.44 (C-50 ), 69.62 (CH2 -Ph), 67.98 (C-1), 38.10 (C-20 ), 25.31 (C-40 ), 25.23 (C-30 ), 15.90 (C-10 ), 14.88 (C-60 ), 8.21 (C-70 ); 13 C NMR (100 MHz, CDCl3) (ca. 16% cis-isomer 21, observable signals) δ 139.22 (C-ipso), 128.43 (C-meta), 127.80 (C-para), 127.44 (C-orto), 70.87 (C-50 ), 69.57 (CH2 -Ph) 67.78 (C-1), 35.64 (C-20 ), 28.43 (C-40 ), 18.93 (C-30 ), 14.19 (C-10 ), 13.37 (C-60 ), 1.98 (C-70 ). HRMS (ESI+) Calcd. for [C15 H20 O2 +H]+ 233.1542, Found: 233.1537. [(10 S,20 R,50 R,60 S)-50 -Hydroxybicyclo [4.1.0]hept-20 -yl]methyl benzoate (23) and its 0 (2 S)-diastereoisomer (24). To a stirred solution of a 2:1 mixture of 21 and 22 (158 mg, 0.68 mmol) in dry CH2 Cl2 (7 mL) at 0 ◦ C, anhydrous Et3 N (100 µL, 0.71 mmol) and benzoyl chloride (91 µL, 0.78 mmol) were sequentially added under argon atmosphere. The mixture was subsequently allowed to attain room temperature overnight. Then, the solution was treated with HCl 10% solution (7 mL) and CH2 Cl2 (7 mL), the two phases were separated, and the aqueous phase was extracted with CH2 Cl2 (3 × 7 mL). The organic layers were Int. J. Mol. Sci. 2022, 23, 9704 11 of 15 washed with brine (30 mL), dried (Na2 SO4 ), and concentrated under reduced pressure to obtain a colorless oil that was directly used for the next step without further purification. Accordingly, the crude was dissolved in EtOH (7 mL) and hydrogenated in the presence of Pd/C (23 mg, 10% wt.) at 2 atm overnight. The mixture was filtered through a short pad of Celite® and washed with additional EtOH. The solvent was evaporated under reduced pressure and purified by column chromatography (hexanes-EtOAc, 10:1 → 5:1) to afford alcohol 23 (104 mg, 0.42 mmol, 62% yield) as a colorless syrup and its isomer 24 (49 mg, 0.20 mmol, 29% yield) as a colorless syrup. 23: [α]D 20 = +48.7 (c 1,02, CHCl3 ); 1 H NMR (400 MHz, CDCl3 ) δ 8.05 (d, Jorto,meta = 7.6 Hz, 2H, H-orto), 7.61–7.51 (m, 1H, H-para), 7.44 (t, Jmeta,orto = Jmeta,para = 7.6 Hz, 2H, H-meta), 4.28 (dd, J1,20 = 6.8 Hz, J1,10 = 2.0 Hz, 2H, H-1), 4.20 (dt, J50 ,40 ax = 9.5 Hz, J50 ,40 eq = J50 ,60 = 5.7 Hz, 1H, H-50 ), 2.0 (dddd, J20 ,30 ax = 13.8 Hz, J20 ,1 = 6.8 Hz, J20 ,30 eq = 4.4 Hz, J20 ,10 = 1.9 Hz, 1H, H-20 ), 1.86–1.72 (m, 1H, H-40 ), 1.70–1.61 (m, 2H, H-30 ), 1.33 (tt, J60 ,70 endo = J60 ,70 exo = 8.8 Hz, J60 ,50 = J60 ,10 = 5.7 Hz, 1H, H-60 ), 1.13–0.87 (m, 2H, H-10 , H-40 ), 0.71 (td, J70 exo,10 = J70 exo,60 = 8.8 Hz, Jgem = 4.9 Hz, 1H, H-70 exo), 0.40 (q, J70 endo,10 = J70 endo,60 = Jgem = 5.3 Hz, 1H, H-70 endo); 13 C NMR (100 MHz, CDCl ) δ 166.8 (C=O), 133.1 (C-para), 130.4 (C-ipso), 129.7 (C-orto), 3 128.5 (C-meta), 69.4 (C-1), 68.0 (C-50 ), 34.5 (C-20 ), 28.0 (C-40 ), 26.0 (C-30 ), 18.1 (C-60 ), 16.2 (C-10 ), 6 (C-70 ). 24: [α]D 20 = −12.3 (c 1.16, CHCl3 ); 1 H NMR (400 MHz, CDCl3 ) δ 8.05 (d, Jorto,meta = 7.2 Hz, 2H, H-orto), 7.56 (t, Jpara,meta = 7.4 Hz, 1H, H-para), 7.44 (t, Jmeta,orto = Jmeta,para = 7.6 Hz, 2H, H-meta), 4.34 (dt, J50 ,40 ax = 7.3 Hz, J50 ,40 eq = J50 ,60 = 4.7 Hz, 1H, H-50 ), 4.29 (dd, Jgem = 10.7 Hz, J1a,20 = 6.9 Hz, 1H, H-1a), 4.18 (dd, Jgem = 10.7 Hz, J1b,20 = 7.3 Hz, 1H, H-1b), 2.36 (dq, J20 ,30 ax = 12.6 Hz, J20 ,1a = J20 ,1b = J20 ,30 eq = 6.7 Hz, 1H, H-20 ), 1.51–1.37 (m, 4H, H-60 , 2H-40 , H-30 ), 1.27 (tt, J10 ,20 = J10 ,70 exo = 8.5 Hz, J10 ,70 endo = J10 ,60 = 5.7 Hz, 1H, H-10 ), 1.23–1.13 (m, 1H, H-30 ), 0.57 (q, Jgem = J70 endo,60 = J70 endo,10 = 5.3 Hz, 1H, H-70 endo), 0.46 (td, J70 exo,10 = J70 exo,60 = 8.9 Hz, Jgem = 5.1 Hz, 1H, H-70 exo); 13 C NMR (100 MHz, CDCl3 ) δ 166.8 (C=O), 133.0 (C-para), 130.6 (C-ipso), 129.7 (C-orto), 128.5 (C-meta), 68.9 (C-1), 64.7 (C-50 ), 31.7 (C-20 ), 29.9 (C-40 ), 19.4 (C-30 ), 17.1 (C-10 ), 14.7 (C-60 ), 1.7 (C-70 ). (1S,2S,5R,6R)-5-[(Benzoyloxy)methyl)]bicyclo [4.1.0]heptan-2-amonium chloride (25). To a stirred solution of Ph3 P (170 mg, 0.65 mmol) in dry toluene (4.5 mL), DBAD (150 mg, 0.65 mmol) was slowly added under argon atmosphere and the mixture was stirred for 45 min at 0 ◦ C. After 15 min, a white suspension appeared. Then, diphenylphosphoryl azide (DPPA, 100 µL, 0.45 mmol) and a solution of 23 (105 mg, 0.43 mmol) in dry toluene (1 mL) were sequentially added. The mixture was allowed to warm to room temperature and stirred overnight. Then, the solvent was removed, and the crude was purified by column chromatography (hexanes-EtOAc, 20:1 to 15:1) to obtain a crude that was dissolved in EtOAc (2 mL) and hydrogenated in the presence of Pd/C (15 mg, 10% wt.) at 2 atm for 24 h. Then, the mixture was filtered through a short pad of Celite® and washed with additional EtOAc. The solvent was evaporated under reduced pressure and the crude was treated with 2 M HCl in Et2 O (2 mL, 4 mmol) at 0 ◦ C, stirred for 2 h, and filtered to furnish the ammonium salt 25 (101 mg, 0.36 mmol, 84% yield) as a brown solid: Mp 130–132 ◦ C (from EtO2 ); [α]D 20 = +14.2 (c 1.0, CHCl3 ); 1 H NMR (400 MHz, CDCl3 ) δ 8.69 (s, 3H, NH3 + ), 8.08–7.97 (m, 2H, H-Ar), 7.56–7.50 (m, 1H, H-Ar), 7.44–7.36 (m, 2H, H-Ar), 4.45 (d, J10 ,5 = 7.7 Hz, 2H, H-10 ), 3.84 (br s, 1H, H-2), 2.15–1.89 (m, 2H, H-5, H-4), 1.60–1.46 (m, 3H, H-3, H-4), 1.31–1.22 (m, 1H, H-1), 1.12–1.02 (m, 1H, H-6), 0.91 (td, J7exo,1 = J7exo,6 = 9.1 Hz, Jgem = 5.1 Hz, 1H, H-7exo), 0.19 (q, Jgem = J7endo,1 = J7endo,6 = 5.5 Hz, 1H, H-7endo); 13 C NMR (100 MHz, CDCl ) δ 166.6 (C=O), 133.1 (C-para), 130.4 (C-ipso), 129.7 (C-orto), 3 128.5 (C-meta), 69.0 (C-10 ), 47.1 (C-2), 33.9 (C-5), 23.2 (C-4), 19.4 (C-3), 14.0 (C-6), 12.5 (C-1), 10.4 (C-7); IR (ATR) ν 3404, 2929, 1712, 1273, 1113, 713 cm−1 . HRMS (ESI+) Calcd. for [C15 H18 NO2 ]+ 244.1338, Found: 244.1334. (E)-3-Ethoxyacryloyl isocyanate (26). Vinyl ether (5.75 mL, 60 mmol) was added dropwise to oxalyl chloride (7.60 mL, 90 mmol) at 0 ◦ C. The reaction mixture was main- tained for 2 h at 0 ◦ C and then warmed to room temperature overnight. Excess oxalyl chloride was distilled off and the black residue was heated at 120 ◦ C for 30 min. Then, Int. J. Mol. Sci. 2022, 23, 9704 12 of 15 the residue was purified by vacuum distillation, using a short Vigreux column, to obtain (2E)-3-ethoxyacryloyl chloride (4.30 g, 31.97 mmol, 53% yield) as a colorless liquid. Silver cyanate (90 mg, 0.60 mmol), previously dried over phosphorus pentoxide at 80 ◦ C for 3 h, in dry benzene (2 mL), was heated to reflux for 30 min and a solution of (2E)-3-ethoxyacryloyl chloride (45 mg, 0.31 mmol) in dry benzene (0.8 mL) was then added dropwise. The mixture was stirred for 30 min before allowing the solid to settle. The supernatant, which is a solution of the isocyanate 25, was then decanted and used directly in the next reaction. [(10 R,20 R,50 S,60 S)-50 -(200 ,400 -dioxo-300 ,400 -dihydropyrimidin-100 (2H)-yl] bicyclo [4.1.0] heptan-20 -yl)methyl benzoate (27). The ammonium chloride 25 (43 mg, 0.153 mmol) was dissolved in dry DMF (1.8 mL), and Et3 N (22 µL, 0.157 mmol) was added. The mixture was cooled to −20 ◦ C and a freshly prepared solution of the isocyanate 26 was added slowly to avoid an increase in the temperature. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated in vacuo, and then water (2 mL) was added. The residue was extracted with EtOAc (2 × 2 mL), washed with brine (2 mL), dried (Na2 SO4 ), filtered, and evaporated under reduced pressure. The residue was dissolved in MeOH (0.40 mL), H2 SO4 (1 M, 0.62 mL) was added, and the mixture was heated to reflux for 3 h. Then, the mixture was concentrated under reduced pressure and purified by column chromatography (CH2Cl2 100% to CH2 Cl2 -MeOH, 20:1) to provide 27 (25 mg, 0.073 mmol, 48% yield) as a yellowish oil: 1 H NMR (400 MHz, MeOH-d4 ) δ 8.05 (d, Jorto,meta = 7.6 Hz, 2H, H-orto), 7.96 (d, J6”,5” = 8.0 Hz, 1H, H-6”), 7.64 (d, Jpara,meta = 7.6 Hz, 1H, H-para), 7.52 (t, Jmeta,orto = Jmeta,para = 7.6 Hz, 2H, H-meta), 5.46 (d, J5”,6” = 8.0 Hz, 1H, H-5”), 4.83 (dt, J50 ,40 ax = 4.3 Hz, J50 ,4eq = J50 ,60 = 2.3 Hz, 1H, H-50 ), 4.45 (d, J1,20 = 6.0 Hz, 2H, H-1), 2.21 (dq, J20 ,30 ax = 11.9 Hz, J20 ,30 eq = J20 1 = 6.0 Hz, 1H, H-20 ), 1.78–1.68 (m, 1H, H-40 eq), 1.54 (tt, Jgem = J40 ax,30 ax = 14.7 Hz, J40 ax,50 = J40 ax,30 eq = 4.3 Hz, 1H, H-40 ax), 1.49–1.40 (m, 1H, H-30 ), 1.32–1.21 (m, 2H, H-30 , H-60 ), 1.12–1.04 (m, 1H, H-10 ), 0.97 (td, J7exo,1 = J7exo,6 = 9.4 Hz, Jgem = 5.0 Hz, 1H, H-70 exo), 0.39 (q, Jgem = J7endo,1 = J7endo,6 = 5.3 Hz, 1H, H-70 endo); 13 C NMR (101 MHz, MeOH-d4 ): δ 168.1 (C=O), 166.3 (C-4”), 152.7 (C-2”), 145.1 (C-6”), 134.4 (C-para), 131.5 (C-ipso), 130.5 (C-orto), 129.7 (C-meta), 101.6 (C-5”), 69.9 (C-1), 52.5 (C-50 ), 34.7 (C-20 ), 24.5 (C-40 ), 20.1 (C-30 ), 15.3 (C-60 ), 13.7 (C-10 ), 10.4 (C-70 ). HRMS (ESI+) Calcd. for [C19 H20 N2 O4 +Na]+ 363.1321, Found: 363.1312. 4-Amino-1-[(10 S,20 S,50 R,60 R)-50 -(hydroxymethyl)bicyclo [4.1.0]hept-20 -yl]pyrimidin- 2(1H)-one (2e). A solution of TsCl (31 mg, 0.16 mmol) in dry CH3 CN (104 µL) was added to a mixture of 27 (27 mg, 0.08 mmol), Et3 N (22 µL, 0.16 mmol), and N-methylpiperidine (12 µL, 0.1 mmol) in dry CH3 CN (135 µL) at 0 ◦ C, and the reaction mixture was stirred for 3 h. Then, 30% NH4 OH was added at 0 ◦ C, and the reaction solution was stirred at room temperature overnight. The mixture was diluted with water (1 mL) and EtOAc (1 mL) and the aqueous phase was extracted with additional CH2 Cl2 (2 × 1 mL). The organic layers were dried (Na2 SO4 ), concentrated under reduced pressure, and purified by column chromatography (CH2 Cl2 -EtOAc, 10:2 → CH2 Cl2 -MeOH 15:1) to provide the protected cytosine analogue as a yellowish oil that was dissolved in a 33% solution of methylamine in EtOH (17 mL) and stirred for 24 h. Then, the mixture was concentrated under reduced pressure and purified by column chromatography (CH2 Cl2 -MeOH, 20:1) to provide cytosine nucleoside analogue 2e (9 mg, 38 µmol, 50% yield) as a yellowish solid: 2e: [α]D 20 = +36.2 (c 0.4, MeOH-d4 ); 1 H NMR (400 MHz, MeOH-d4 ) δ 8.04 (d, J6,5 = 7.4 Hz, 1H, H-6), 5.90 (d, J5,6 = 7.4 Hz, 1H, H-5), 4.91–4.87 (m, 1H, H-20 ) 3.64 (dd, Jgem = 10.7 Hz, J1”a,50 = 5.9 Hz, 1H, H-1”a), 3.59 (dd, Jgem = 10.7 Hz, J1”a,50 = 5.9 Hz, 1H, H-1”b), 1.85 (dq, J50 ,40 ax = 11.4 Hz, J50 40 eq = J50 ,1”a = J50 ,1”b = 5.9 Hz, 1H, H-50 ), 1.68 (dt, Jgem = 13.7 Hz, J30 eq,40 ax = J30 eq,50 = 4.4 Hz, 1H, H-30 eq), 1.48 (tt, Jgem = J30 ax,40 ax = 13.7 Hz, J30 ax, 40 eq = J30 ax,50 = 3.6 Hz, 1H, H-30 ax), 1.34–1.26 (m, 2H, H-10 , H-40 a), 1.19–1.07 (m, 1H, H-40 b), 1.00 (td, J60 ,70 exo = J60 ,10 = 9.4 Hz, J60 ,70 endo = 5.2 Hz, 1H, H-60 ), 0.89 (td, J7exo,1 = J7exo,6 = 9.4, Jgem = 5.2 Hz, 1H, H-70 exo), 0.30 (q, Jgem = J7endo,1 = J7endo,6 = 5.2 Hz, 1H, H-70 endo); 13 C NMR (100 MHz, MeOH-d4 ): δ 167.3 (C-4), 159.0 (C-2), 145.6 (C-6), 95.0 (C-5), 67.6 (C-1”), 52.8 (C-20 ), 37.5 Int. J. Mol. Sci. 2022, 23, 9704 13 of 15 (C-50 ), 24.6 (C-30 ), 19.6 (C-40 ), 15.7 (C-10 ), 13.81 (C-60 ), 10.26 (C-70 ). HRMS (ESI+) Calcd. for [C12 H17 N3 O2 +H]+ 236.1399, Found: 236.1394. 4. Conclusions In this article, an easy route to enantiomerically pure 4-hydroxy-2-cyclohexanone derivatives 1a,b, which are commonly used as chiral building blocks, was identified on the basis of ATH with bifunctional ruthenium catalysts. This approach provided convenient access to both enantiomers from the same precursor by selecting either the (S,S)- or (R,R)-5 catalyst. In addition, a stereoselective route to the cytosine analogue, built on a bicyclo [4.1.0]heptane scaffold, was finely tuned, starting from cyclohexanol (R)-6. Finally, the kinase activity assays of compounds 2a–e showed that compounds 2b and 2d display affinity for HSV-1 TK but not for human TK-1, thus validating our previous molecular modeling study and pointing to their selectivity for herpetic TKs but not cellular TKs. Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms23179704/s1. Author Contributions: S.J. and B.D.-P. contributed equally to this article; F.B. and R.A. devised the project’s concept and designed the experiments; S.J. and B.D.-P. synthesized compounds, super- vised by O.I., F.B.; and R.A.; J.B. performed the biological assays; R.A. wrote the manuscript with contributions of the other authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Economy, Industry, and Competi- tiveness (MINECO) (projects: CTQ2016-75363-R and PID2019-106403RB-I00), co-financed with the European Fund for Regional Development (FEDER). Acknowledgments: We acknowledge the support of Generalitat de Catalunya (2017-SGR-191). We thank Lizette van Berckelaer and Ria van Berwaer for their excellent technical assistance. S.J. and B.D.-P. acknowledge the financial support provided by the Universitat Autònoma de Barcelona and Generalitat de Catalunya, respectively. Conflicts of Interest: The authors declare no conflict of interest. References 1. Huber, T.; Preuhs, T.A.; Gerlinger, C.K.G.; Magauer, T. Development of a β-C−H Bromination Approach toward the Synthesis of Jerantinine E. J. Org. Chem. 2017, 82, 7410–7419. [PubMed] 2. White, J.D.; Li, Y.; Kim, J.; Terienk, M. A Novel Synthesis of (-)-Huperzine A via Tandem Intramolecular Aza-Prins Cyclization- Cyclobutane Fragmentation. Org. Lett. 2013, 15, 882–885. [PubMed] 3. Kawasumi, M.; Kanoh, N.; Iwabuchi, Y. Concise Entry to Both Enantiomers of 8-Oxabicyclo[3.2.1]oct-3-en-2-one Based on Novel Oxidative Etherification: Formal Synthesis of (-)-Sundiversifolide. Org. Lett. 2011, 13, 3620–3623. [PubMed] 4. Hughes, C.C.; Miller, A.K.; Trauner, D. An Electrochemical Approach to the Guanacastepenes. Org. Lett. 2005, 7, 3425–3428. 5. Wehlauch, R.; Grendelmeier, S.M.; Miyatake-Ondozabal, H.; Sandtorv, A.H.; Scherer, M.; Gademann, K. Investigating Biogenetic Hypotheses of the Securinega Alkaloids: Enantioselective Total Syntheses of Secu’amamine E/ent-Virosine A and Bubbialine. Org. Lett. 2017, 19, 548–551. 6. Kawashima, H.; Sakai, M.; Kaneko, Y.; Kobayashi, Y. Further Study on Synthesis of the Cyclobakuchiols. Tetrahedron 2015, 71, 2387–2392. 7. Burns, A.R.; Taylor, R.J.K. Synthetic Approaches to Enantiomerically Enriched 4-Hydroxycyclohex-2-en-1-one—A Key Chiral Building Block in Complex Natural Product Synthesis. Synthesis 2011, 2011, 681–707. 8. Hua, Z.; Yu, W.; Su, M.; Jin, Z. Synthetic Studies toward the Construction of the cis-Decalin Portion of Superstolides A and B. Application of a Sequential Double Michael Reaction and an Anionic Oxy-Cope Rearrangement. Org. Lett. 2005, 7, 1939–1942. 9. Bayón, P.; Marjanet, G.; Toribio, G.; Alibés, R.; De March, P.; Figueredo, M.; Font, J. An Efficient Protocol for the Enantioselective Preparation of a Key Polyfunctionalized Cyclohexane. New Access to (R)- and (S)-4-Hydroxy-2-cyclohexenone and (R)- and (S)-trans-Cyclohex-2-ene-1,4-diol. J. Org. Chem. 2008, 73, 3486–3491. 10. Fudickar, W.; Vorndran, K.; Linker, T. Auxiliary Controlled Singlet-Oxygen Ene Reactions of Cyclohexenes. Tetrahedron 2006, 62, 10639–10646. 11. Trost, B.M.; Masters, J.T.; Lumb, J.-P.; Fateen, D. Asymmetric Synthesis of Chiral Cycloalkenone Derivatives via Palladium Catalysis. Chem. Sci. 2014, 5, 1354–1360. [CrossRef] [PubMed] Int. J. Mol. Sci. 2022, 23, 9704 14 of 15 12. Arthurs, C.L.; Morris, G.A.; Piacenti, M.; Pritchard, R.G.; Stratford, I.J.; Tatic, T.; Whitehead, R.C.; Williams, K.F.; Wind, N.S. The Synthesis of 2-Oxyalkyl-cyclohex-2-enones, Related to the Bioactive Natural Products COTC and Antheminone A, which Possess Anti-Tumour Properties. Tetrahedron 2010, 66, 9049–9060. [CrossRef] 13. Audia, J.E.; Boisvert, L.; Patten, A.D.; Villalobos, A.; Danishefsky, S.J. Synthesis of Two Useful, Enantiomerically Pure Derivatives of (S)-4-Hydroxy-2-cyclohexenone. J. Org. Chem. 1989, 8, 3738–3740. [CrossRef] 14. Domínguez-Pérez, B.; Ferrer, E.; Figueredo, M.; Maréchal, J.D.; Balzarini, J.; Alibés, R.; Busqué, F. Synthesis of Novel Nucleoside Analogues Built on a Bicyclo[4.1.0]heptane Scaffold. J. Org. Chem. 2015, 80, 9495–9505. [CrossRef] 15. Eyer, L.; Nencka, R.; De Clercq, E.; Seley-Radtke, K.; Růžek, D. Nucleoside Analogs as a Rich Source of Antiviral Agents Active Against Arthropod-Borne Flaviviruses. Antivir. Chem. Chemother. 2018, 26, 1–28. [CrossRef] 16. Toribio, G.; Marjanet, G.; Alibés, R.; de March, P.; Font, J.; Bayón, P.; Figueredo, M. Divergent Approach to Gabosines and Anhydrogabosines: Enantioselective Syntheses of (+)-Epiepoformin, (+)-EpoKformin, (+)-Gabosine A, and Gabosines B and F. Eur. J. Org. Chem. 2011, 2011, 1534–1543. [CrossRef] 17. Ferrer, E.; Alibés, R.; Busqué, F.; Figueredo, M.; Font, J.; de March, P. Enantiodivergent Synthesis of Cyclohexenyl Nucleosides. J. Org. Chem. 2009, 74, 2425–2432. [CrossRef] 18. Bardaji, G.G.; Cantó, M.; Alibés, R.; Bayón, P.; Busqué, F.; de March, P.; Figueredo, M.; Font, J. Diastereoselective Synthesis of Allosecurinine and Viroallosecurinine from Menisdaurilide. J. Org. Chem. 2008, 73, 7657–7662. [CrossRef] 19. Noyori, R.; Hashiguchi, S. Asymmetric Transfer Hydrogenation Catalyzed by Chiral Ruthenium Complexes. Acc. Chem. Res. 1997, 30, 97–102. [CrossRef] 20. Wang, D.; Astruc, D. The Golden Age of Transfer Hydrogenation. Chem. Rev. 2015, 115, 6621–6686. [CrossRef] 21. Foubelo, F.; Nájera, C.; Yus, M. Catalytic Asymmetric Transfer Hydrogenation of Ketones: Recent Advances. Tetrahedron: Asymmetry 2015, 26, 769–790. [CrossRef] 22. Ferrie, L.; Boulard, L.; Pradaux, F.; Bouzbouz, S.; Capdevielle, P.; Cossy, J. Synthetic Efforts toward the Macrolactone Core of Leucascandrolide A. J. Org. Chem. 2008, 73, 1864–1880. [CrossRef] [PubMed] 23. Matsumura, K.; Hashiguchi, S.; Ikariya, T.; Noyori, R. Asymmetric Transfer Hydrogenation of α, β-Acetylenic Ketones. J. Am. Chem. Soc. 1997, 119, 8738–8739. [CrossRef] 24. Fujii, A.; Hashiguchi, S.; Uematsu, N.; Ikariya, T.; Noyori, R. Ruthenium(II)-Catalyzed Asymmetric Transfer Hydrogenation of Ketones Using a Formic Acid-Triethylamine Mixture. J. Am. Chem. Soc. 1996, 118, 2521–2522. [CrossRef] 25. Wu, J.; Wang, F.; Ma, Y.; Cui, X.; Cun, L.; Zhu, J.; Deng, J.; Yu, B. Asymmetric Transfer Hydrogenation of Imines and Iminiums Catalyzed by a Water-Soluble Catalyst in Water. Chem. Commun. 2006, 1766–1768. [CrossRef] 26. Holub, N.; Neidhöfer, J.; Blechert, S. Total Synthesis of (+)-trans-195A. Org. Lett. 2005, 7, 1227–1229. [CrossRef] 27. Xu, B.; Xun, W.; Wang, T.; Qiu, F.G. Total Synthesis of (+)-Aplykurodinone-1. Org. Lett. 2017, 19, 4861–4863. [CrossRef] 28. Teitelbaum, A.M.; Meissner, A.; Harding, R.A.; Wong, C.A.; Remmel, R.P. Synthesis, Ph-Dependent, and Plasma Stability of Meropenem Prodrugs for Potential Use Against Drug-Resistant Tuberculosis. Bioorg. Med. Chem. 2013, 21, 5605–5617. [CrossRef] 29. Lebel, H.; Marcoux, J.F.; Molinaro, C.; Charette, A.B. Stereoselective cyclopropanation reactions. Chem. Rev. 2003, 103, 977–1050. [CrossRef] 30. Yang, Z.; Lorenz, J.C.; Shi, Y. Exploring New Reactive Species for Cyclopropanation. Tetrahedron Lett. 1998, 39, 8621–8624. [CrossRef] 31. Kim, H.Y.; Walsh, P. Efficient Approaches to the Stereoselective Synthesis of Cyclopropyl Alcohols. Acc. Chem. Res. 2012, 45, 1533–1547. [CrossRef] [PubMed] 32. Furukawa, J.; Kawabata, N.; Nishimura, J. Synthesis of Cyclopropanes by The Reaction of Olefins with Dialkylzinc and Methylene Iodide. Tetrahedron Lett. 1968, 24, 53–58. [CrossRef] 33. Scott, J.P.; Alam, M.; Bremeyer, N.; Goodyear, A.; Lam, T.; Wilson, R.D.; Zhou, G. Inversion of a Secondary Alcohol with Diphenylphosphoryl azide. Application to the Enantioselective Multikilogram Synthesis of a HCV Polymerase Inhibitor. Org. Process Res. Dev. 2011, 15, 1116–1123. [CrossRef] 34. Fernández, F.; García-Mera, X.; Morales, M.; Rodríguez-Borges, J.E. A Short, Efficient Synthesis of Substituted Uracil: An Indane Carbocyclic Nucleoside. Synthesis 2001, 2, 239–242. [CrossRef] 35. Komatsu, H.; Morizane, K.; Kohno, T.; Tanikawa, H. An Efficient Amination Method for Manufacturing Cytidines. Org. Process Res. Dev. 2004, 8, 564–567. [CrossRef] 36. Balzarini, J.; Van Daele, I.; Negri, N.; Solaroli, N.; Karlsson, A.; Liekens, S.; Gago, F.; Van Calenbergh, S. Human Mitochondrial Thymidine Kinase Is Selectively Inhibited by 30 -Thiourea Derivatives of β-Thymidine: Identification of Residues Crucial for Both Inhibition and Catalytic Activity. Mol. Pharmacol. 2009, 75, 1127–1136. [CrossRef] 37. Ren, K.; Zhao, M.; Hu, B.; Xie, X.; Ratovelomamana-Vidal, V.; Zhang, Z. An Enantioselective Approach to 4-O-Protected-2- Cyclopentene-1,4-Diol Derivatives via Rhodium-Catalyzed Redox-Isomerization Reaction. J. Org. Chem. 2015, 80, 12572–12579. [CrossRef] 38. Tachihara, T.; Kitahara, T. Total Synthesis of (1)-Epiepoformin, (1)-Epiepoxydon and (1)-Bromoxone Employing a Useful Chiral Building Block, Ethyl (1R,2S)-5,5-Ethylenedioxy-2-hydroxycyclohexanecarboxylate. Tetrahedron 2003, 59, 1773–1780. [CrossRef] 39. Matsuzawa, M.; Kayeka, H.; Yamaguchi, J.; Shoji, M.; Onose, R.; Osada, H.; Hayashi, Y. Enantio- and Diastereoselective Total Synthesis of (+)-Panepophenanthrin, a Ubiquitin-Activating Enzyme Inhibitor, and Biological Properties of Its New Derivatives. Chem. Asian J. 2006, 1, 845–851. [CrossRef] Int. J. Mol. Sci. 2022, 23, 9704 15 of 15 40. Touge, T.; Hakamata, T.; Nara, H.; Kobayashi, T.; Sayo, N.; Saito, T.; Kayaki, Y.; Ikariya, T. Oxo-tethered Ruthenium(II) Complex as a Bifunctional Catalyst for Asymmetric Transfer Hydrogenation and H2 -Hydrogenation. J. Am. Chem. Soc. 2011, 133, 14960–14963. [CrossRef] 41. Kitahara, T.; Kurata, H.; Mori, K. Efficient Synthesis of The Natural Enantiomer of Sporogen-AO 1 (13-Desoxyphomenone) a Sporogenic Sesquiterpene from Aspergillus Oryzae. Tetrahedron 1988, 44, 4339–4349. [CrossRef]